人血管抑素基因分泌型融合载体的构建与表达

Construction and expression of a secretory-fusion plasmid containing human angiostatin gene

【目的】构建人血管抑素(angiostatin,As)原核分泌型融合表达载体pEZZ18-As,诱导表达As融合蛋白。【方法】根据人AscDNA序列的开放读码框架设计上、下游引物,从人肝组织中提取总RNA后,利用RT-PCR技术扩增As基因;酶切目的基因与pEZZ18载体,回收纯化后做定向克隆连接,转化E.coliDH5α;XbaⅠ及EcoRⅠ双酶切电泳与测序鉴定pEZZ18-As;诱导培养重组菌,表达目的蛋白。【结果】酶切pEZZ18-As,电泳分析插入片段长度为1111bp,与预期结果一致,对连接点两端进行测序,结果显示接头两端序列连接正确;SDS-PAGE分析诱导培养重组菌,新出现的蛋白大小约61kD,与预期相符。【结论】成功构建人血管抑素表达载体并表达出目的蛋白,该研究为抗血管药物治疗实体瘤奠定了基础。

To clone human angiostatin gene into a secretory-fusion vector, then to express.The primers were designed according to the open reading frame of angiostatin gene cDNA. The total RNA was isolated from human hepatocytes. The angiostatin gene was amplified from the mRNA by RT-PCR and the PCR products was cloned into pEZZ18 vector, then to express in E.coli DH5α.Restricted enzymes analysis and DNA sequencing showed that the sequence of the recombinant plasmid pEZZ18-As was correct. SDS-PAGE analysis testified that the fusion angiostatin came up.[Conclusion]The angiostatin gene was successfully cloned and there was expression of the fusion protein in the recombinant E.coli DH5α.The experiment lays a foundation for tumor-antiangiogenesis drug therapy.